Delineating the core regulatory elements crucial for directed cell migration by examining folic-acid-mediated responses

Dictyostelium discoideum shows chemotaxis towards folic acid (FA) throughout vegetative growth, and towards cAMP during development. We determined the spatiotemporal localization of cytoskeletal and signaling molecules and investigated the FA-mediated responses in a number of signaling mutants to further our understanding of the core regulatory elements that are crucial for cell migration. Proteins enriched in the pseudopods during chemotaxis also relocalize transiently to the plasma membrane during uniform FA stimulation. In contrast, proteins that are absent from the pseudopods during migration redistribute transiently from the PM to the cytosol when cells are globally stimulated with FA. These chemotactic responses to FA were also examined in cells lacking the GTPases Ras C and G. Although Ras and phosphoinositide 3-kinase activity were significantly decreased in Ras G and Ras C/G nulls, these mutants still migrated towards FA, indicating that other pathways must support FA-mediated chemotaxis. We also examined the spatial movements of PTEN in response to uniform FA and cAMP stimulation in phospholipase C (PLC) null cells. The lack of PLC strongly influences the localization of PTEN in response to FA, but not cAMP. In addition, we compared the gradient-sensing behavior of polarized cells migrating towards cAMP to that of unpolarized cells migrating towards FA. The majority of polarized cells make U-turns when the cAMP gradient is switched from the front of the cell to the rear. Conversely, unpolarized cells immediately extend pseudopods towards the new FA source. We also observed that plasma membrane phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] levels oscillate in unpolarized cells treated with Latrunculin-A, whereas polarized cells had stable plasma membrane PtdIns(3,4,5)P(3) responses toward the chemoattractant gradient source. Results were similar for cells that were starved for 4 hours, with a mixture of polarized and unpolarized cells responding to cAMP. Taken together, these findings suggest that similar components control gradient sensing during FA- and cAMP-mediated motility, but the response of polarized cells is more stable, which ultimately helps maintain their directionality

Microsc Microanal

Abstract A microcompressor is a precision mechanical device that flattens and immobilizes living cells and small organisms for optical microscopy, allowing enhanced visualization of sub-cellular structures and organelles. We have developed an easily fabricated device, which can be equipped with microfluidics, permitting the addition of media or chemicals during observation. This device can be used on both upright and inverted microscopes. The apparatus permits micrometer precision flattening for nondestructive immobilization of specimens as small as a bacterium, while also accommodating larger specimens, such as Caenorhabditis elegans, for long-term observations. The compressor mount is removable and allows easy specimen addition and recovery for later observation. Several customized specimen beds can be incorporated into the base. To demonstrate the capabilities of the device, we have imaged numerous cellular events in several protozoan species, in yeast cells, and in Drosophila melanogaster embryos. We have been able to documen

The Conjusome—A Transient Organelle Linking Genome Rearrangements in the Parental and Developing Macronuclei

The conjusome plays an important role in the conjugation events that occur in Tetrahymena thermophila. The conjusome appears in the anterior of conjugant pairs during the early stages of new macronuclei (anlagen) development. It lacks a membrane, and is composed of a network of fibrous, electron dense material, containing background cytoplasm and ribosomes. Several proteins localize to this organelle, including Pdd1p, a chromodomain protein that participates in the formation of chromatin-containing structures in developing macronuclear anlagen, and is associated with the elimination of specific germ-line sequences from developing macronuclei. Conjugants lacking the PDD1 allele in the parental macronucleus do not show Pdd1p antibody staining in conjusomes. Investigations were performed using mutant cell lines, uniparental cytogamy and drug treatment, and show that the conjusome appears to be dependent on parental macronuclei condensation, and is a transitory organelle that traffics nuclear determinants from the parental macronucleus to the developing anlagen. These data, taken together with Pdd1p knockout experiments, suggest the conjusome is involved in the epigenetic phenomena that occur during conjugation and sexual reorganization. This is likely a conserved organelle. Conjusome-like structures were also observed in another Ciliate, Stylonichia. In general, conjusomes have features that resemble germ line P-granules

The Conjusome—A Transient Organelle Linking Genome Rearrangements in the Parental and Developing Macronuclei

The conjusome plays an important role in the conjugation events that occur in Tetrahymena thermophila. The conjusome appears in the anterior of conjugant pairs during the early stages of new macronuclei (anlagen) development. It lacks a membrane, and is composed of a network of fibrous, electron dense material, containing background cytoplasm and ribosomes. Several proteins localize to this organelle, including Pdd1p, a chromodomain protein that participates in the formation of chromatin-containing structures in developing macronuclear anlagen, and is associated with the elimination of specific germ-line sequences from developing macronuclei. Conjugants lacking the PDD1 allele in the parental macronucleus do not show Pdd1p antibody staining in conjusomes. Investigations were performed using mutant cell lines, uniparental cytogamy and drug treatment, and show that the conjusome appears to be dependent on parental macronuclei condensation, and is a transitory organelle that traffics nuclear determinants from the parental macronucleus to the developing anlagen. These data, taken together with Pdd1p knockout experiments, suggest the conjusome is involved in the epigenetic phenomena that occur during conjugation and sexual reorganization. This is likely a conserved organelle. Conjusome-like structures were also observed in another Ciliate, Stylonichia. In general, conjusomes have features that resemble germ line P-granules

Dietary analysis of Piraino 1, Sicily, Italy: The role of archaeopalynology in forensic science

Pollen from intestinal contents of mummies, backed by macrofloral analysis, provides important clues to diet, medicines, and season of death. Intestinal contents were recovered from the Piraino 1 mummy from the “Sepulcher of the Priests,” Piraino Mother Church, in the province of Messina, Sicily. Using standard palynological methods and pollen concentration technique, we quantified the numbers of pollen grains per gram of coprolite. The pollen spectrum was dominated by Polygalaceae, the Milkwort Family. Polygalaceae pollen is rarely found in archaeological sites. Based on comparison to published keys, we determined that the pollen came from a species of Polygala. Polygala contains species with medicinal value. We found nine other pollen types. Traces of Potamogeton (pondweed) was observed in preliminary scans. Multiple grains of Brassicaceae (mustard family), cereal (cultivated grains), Typha (cattail) and Cheno-am were found. Single grains of Castanea (chestnut), Fabaceae (bean family), Salix (willow), and Solanaceae (tomato family) were found. The preservation of the pollen was poor except for Polygala and the cereal pollen. Brassicaceae and Cheno-am have been part of the natural pollen spectrum in Sicily since ancient times. Cereal grains were consumed with prepared food. Importantly, background arboreal pollen was nearly absent. This indicates that Piraino 1 died during months of low pollination. The absence of olive pollen is important since this plant in Sicily reaches its maximum pollination in May and June and tapers off rapidly by mid-June. Therefore, absence of the key warm season airborne pollen type suggests a post-June death. Macrofossils, especially residue from grape pulp, indicates a death in September to November. His cause of death was likely a result of multiple myeloma. The methodological differences between archaeopalynology and forensic palynology are summarized. We suggest that the palynological methods presented here should be adopted for human remains analysis in forensic palynology